R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co-transcriptionally in the opposite orientation to replication fork progression. A recent study has shown that replication forks stalled by co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by ELL-dependent reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds R-loops in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4-ELL pathway for resolution of R-loop-mediated transcription-replication conflicts, which may be involved in R-loop unwinding.

Centre for Drug Discovery and Development Sathyabama Institute of Science and Technology Chennai 600119 India

Faculty of Science Charles University Prague Albertov 6 128 00 Prague 2 Czech Republic

Institute of Molecular Cancer Research University of Zurich Winterthurerstrasse 190 8057 Zurich Switzerland

Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

School of Life Sciences University of Bedfordshire Park Square Luton LU1 3JU UK

School of Life Sciences University of Westminster 115 New Cavendish Street London W1W 6UW UK

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